The development and research of antigen binding moieties is a field which rapidly has processed within the last 30 years. Meanwhile particularly monoclonal antibodies are well established as biotherpeutic agents but also other scaffolds comprising target specificity already entered the market or proceed in development. Apart from that the use of antibodies and fragments thereof are self evident and indispensable for research purposes.
For the selection of antigen binding moieties a wide range of different techniques are in use. Apart of the isolation of antibody-producing hybridoma from immunized mammals the use of synthetic libraries for phage or yeast display are only few of many examples.
However, the selection process to isolate the target-specific candidates from a highly diverse library is a crucial step within any antibody generation project and therefor many different strategies and procedures are well established. Generally, the selection process comprises the incubation of the library with the antigen and the subsequent isolation of antibody-antigen complexes. Thereby the antigen can be presented to the library in solution, immobilized on a solid phase or presented on a cell. However in order to select antigen binding moieties which are directed to a specific epitope, domain or site of an antigen these strategies are limited and desired candidates are isolated only by chance and thus have to be identified during extensive experimental characterization.
However in certain cases a binding moiety which detects only a specific variant of an antigen or a specific relevant epitope is necessary. Thereby the specificity of the antibody shall be directed to for example specific isoforms or splice variants of antigens or only to monomeric or heteromeric compositions of the target. Furthermore the detection of an active form but not the inactive form of an enzyme states another scenario which necessitates an epitope or site-directed selection.
Antibodies targeting disease-relevant enzymes are not only relevant for research purposes but also for use as a therapeutic agent or diagnostic tool for many indications. For example the antagonism of proteases can be used to inhibit uncontrollable bleeding during surgery exemplified by the development of the trypsin inhibitor Aprotinin but also Kallikrein Inhibitors that were described to be potentially useful not only to reduce blood loss during surgery but also to treat allery mediated hereditary angioedema. Furthermore sectreted proteases from pathogens provide additional potential targets for therapeutic but also diagnostic antibodies.
A specific example is the NS2B-NS3 proteinase (NS2B-NS3pro) expressed by the West Nile virus (WNV) which is a virus of the family Flaviviridae. It mainly infects birds, but is known to infect humans, horses, dogs, cats, bats, chipmunks, skunks, squirrels, and domestic rabbits. The main route of human infection is through the bite of an infected mosquito. WNV may have different effects on humans—asymptomatic infection; a mild febrile syndrome termed West Nile Fever; or a neuroinvasive disease termed West Nile meningitis or encephalitis.
2007, in the United States 2007 there were a total of 3,630 cases of WNV neuroinvasive disease (WNND) and 124 deaths were reported (MMWR Morb. Mortal. Wkly. Rep. 57 (26): 720-3. July 2008). 3.4% of the serious infections of WNV were fatal.
WNV control is largely achieved through mosquito control, by elimination of mosquito breeding sites, larviciding active breeding areas and encouraging personal use of mosquito repellents. Along with such efforts go environmental concerns and questions whether the detrimental health effects of spraying pesticides outweigh the relatively few lives which may be saved.
There is no specific treatment for West Nile virus infection. Intensive supportive therapy is directed toward the complications of brain infections. Anti-inflammatory medications, intravenous fluids, and intensive medical monitoring may be required in severe cases. There is no specific antibiotic or antidote for the viral infection. There is also no vaccine to prevent the virus. Amongst the therapeutics under investigation is AMD 3100 (Plerixafor, Genzyme, Inc.), a small organic compound which has been proposed as an antiretroviral drug for HIV, and morpholino antisense oligonucleotides conjugated to cell penetrating peptides (AVI BioPharma, Inc.) There have also been attempts to treat infections using ribavirin, intravenous immunoglobulin, or alpha interferon, and it has been found that blocking angiotensin II can treat the “cytokine storm” induced by WNV (Curr Top Med Chem 4 (13): 1433-54). We are however still far from an effective therapy for this emerging virus.
WNV is an enveloped, positive-stranded, 11-kb RNA virus. The genomic RNA of WNV encodes a polyprotein precursor which consists of three structural proteins (C, capsid; prM, membrane, and E, envelope) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) arranged in the order C-prM-E-NS1-NS2A-NS2BNS3-NS4A-NS4B-NS5.
Polyprotein processing by the viral two component NS2B-NS3 proteinase (NS2B-NS3pro) and also by the host cell secretase and furin is required to generate individual viral proteins. The full-length NS3 is a multifunctional protein in which the N-terminal 184 amino acid residues represent the NS3pro domain and the C-terminal sequence codes for the enzyme with the helicase, nucleoside triphosphatase and RNA triphosphatase activities, all of which are coordinately regulated through localization within membrane compartments in the infected cell. NS2B functions as an essential cofactor of NS3pro. The cofactor activity of the 48 amino acid central portion of NS2B is roughly equivalent to that of the entire NS2B sequence (Biochem J. 401, 743-752). Structural studies have determined that NS2B wraps around NS3pro, completing, in a precise and well-defined fashion, the structure of the active site. In agreement, deletion of the NS2B sequence inactivates the functional activity of NS3pro (J. Virol. 78, 13708-13716).
NS3pro is responsible for the cleavage of the capsid protein C and at the NS2A/NS2B, NS2B/NS3, NS3/NS4A, NS4A/NS4B and NS4B/NS5 boundaries. Because inactivating mutations of the NS3pro cleavage sites in the polyprotein precursor abolished viral infectivity (J. Virol. 67, 6797-6807; Curr Med. Chem. 15, 2771-2784) it is a reasonable expectation that the NS3pro function is vitally important for the virus, and that NS3pro antagonists have may have merit as viral drugs.
Several peptide and small organic inhibitors of NS3pro have recently been identified (PLoS Negl Trop Dis. 3, e356; Assay Drug Dev Technol. 5, 737-750; J Med. Chem. 49, 6585-6590; Antiviral Res. 80, 94-101; J Gen Virol. 88, 2223-2227; Antimicrob Agents Chemother. 52, 3385-3393; J Med. Chem. 51, 5714-5721; Antiviral Res. 82, 110-114). There are, however, obstacles which all of these molecules, e.g. inefficient cell penetration, poor solubility and lack of stability.
Antibodies could provide a much sought, excellent scaffold for designing inhibitors targeted to this enzyme. Over the past decade recombinant technology has enabled the production of engineered antibody fragments such as Fabs or single-chain FV (scFv) fragments and phage display technology has been used successfully for the isolation of specific scFv or Fab from human repertoire libraries (Curr. Opin. Biotechnol. 13, 598-602). For phage display selection, the Fab format is preferred since scFv fragments have a high tendency to form multimers. Fab are more stable compared to scFv fragments and tend to stay completely monomeric, allowing selection for affinity in contrast to selection for avidity.
In the present application we describe a novel screening strategy which enables the selection of antigen binding moieties that target only a specific epitope, site or domain of an antigen. To exemplify the effectiveness of the claimed selection strategy we describe the first successful generation of antibodies against WNV NS2B-NS3pro which are highly selective and target the active-site of WNV NS2B-NS3pro. Various uses and analysis upon application of the antibodies of the present invention are disclosed herein below. The generation of antibodies or antibody fragments which are directed only to the active variant of an enzyme enables the development of more efficient therapeutic agents. The specificity only for the variant which is therapeutically relevant facilitates the development of highly efficient antibodies with reduced side effects due to lacking cross-reactivity to the inactive form accompanied by the advantage that lower doses have to be administered. Additionally the disclosed screening method dismisses all antibodies targeting an irrelevant epitope and therby accelerates lead candidate identification and development. Furthermore the antibodies that bind specifically to the active site of a protein isolated by the disclosed screening method can be used for the identification of idiotypic antibodies which mimic the activity of the enzyme/protein. In turn such antibodies bearing enzymatic activity can be used for therapeutic purposes.